O R I G I N A L P A P E R
Development of Mice with Brain-Specific Deletion of Floxed Glud1
(Glutamate Dehydrogenase 1) Using Cre Recombinase Driven
by the Nestin Promoter
Melis Karaca•Pierre Maechler
Received: 21 December 2012 / Revised: 8 April 2013 / Accepted: 9 April 2013 / Published online: 18 April 2013 Ó Springer Science+Business Media New York 2013
Abstract In the brain, Glud1-encoded glutamate dehy-drogenase plays a major role in the recycling of the neu-rotransmitter glutamate. We recently reported a new model of brain-specific Glud1 null mice (Cns-Glud1-/-) lacking glutamate dehydrogenase in the central nervous system. Cns-Glud1-/- mice exhibit reduced astrocytic glutamate breakdown and redirection of glutamate pathways without altering synaptic transmission. Cns-Glud1-/- mice were generated using LoxP and Cre technology. Nestin-Cre mice are widely used to investigate gene deletion in the central nervous system. However, the Nes-Cre transgene itself was reported to induce a phenotype related to body weight gain. Here, we review the potential side-effects of Nes-Cre and analysed Cns-Glud1-/- body weight gain. Overall, Nestin-Cre mice may exhibit transient and mod-erate growth retardation during the few weeks immediately following weaning. Pending appropriate controls and homogenization of the genetic background, Nestin-Cre technology is a valuable tool enabling disruption of genes of interest in the central nervous system.
Keywords Glutamate dehydrogenase Glud1 Brain Nestin-Cre
Glucose is the major source of energy delivered to the central nervous system (CNS) by peripheral organs. However,
within the brain, oxidative catabolism of the neurotrans-mitter glutamate might significantly contribute to ATP pro-duction and maintenance of energy homeostasis. Upon glutamate transmission, intersynaptic glutamate clearance is achieved mostly by astrocytes [1]. Active astrocytic inter-nalization of glutamate also ensures detoxification of the extracellular space. However, altered glutamate handling is associated with various neurodegenerative diseases; such as Parkinson’s disease, epilepsy, schizophrenia, and Alzhei-mer’s disease [2]. Following its uptake by astrocytes gluta-mate may be amidated to glutamine and then recycled back to glutamate once transferred to neurons (Fig. 1a). Alterna-tively, astrocytic glutamate may be deaminated to a-keto-glutarate within mitochondria. This pathway is catalysed either by transaminases or by glutamate dehydrogenase (GDH) before oxidative catabolism in the TCA cycle, thereby promoting ATP generation [3]. In the CNS, GDH is predominantly expressed in astrocytes and to lower levels in neurons [4,5]. GDH catalyses either the anabolic amination of a-ketoglutarate to glutamate or the catabolic oxidation of glutamate [3]. The enzyme forms homohexamers in mito-chondria and is encoded by Glud1 in all vertebrates [6]. Additionally, a second isoform is found in humans [7], being expressed mostly in astrocytes and testicular supporting cells [8].
We recently reported a new model of brain-specific Glud1 null mice (Cns-Glud1-/-) lacking GDH in the CNS [9]. In astrocytes lacking GDH, oxidative catabolism of glutamate to CO2 is reduced, indicating lower astrocytic
glutamate breakdown. However, brain-targeted NMR measurements have shown that Cns-Glud1-/- glutamate levels are maintained to control values, while glutamine concentrations are increased in Cns-Glud1-/- brains [9]. This modified pattern of glutamate/glutamine ratio does not induce over-excitability or hypo-excitability. These data
Special Issue: In honor of Andreas Plaitakis. M. Karaca P. Maechler (&)
Department of Cell Physiology and Metabolism, University of Geneva Medical Centre, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland
e-mail: [email protected]
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Neurochem Res (2014) 39:456–459 DOI 10.1007/s11064-013-1041-0
suggest preservation of glutamate homeostasis in GDH-null brains, at the expense of an increase in glutamine formation. Redirection of glutamate pathways in Cns-Glud1-/-brains is apparently favoured by up-regulation of astrocyte-type glutamate and glutamine transporters and of glutamine synthetase (Fig.1b and [9]). The Cns-Glud1 -/-mouse model represents a unique tool to explore the importance of GDH as a key enzyme connecting glucose and glutamate metabolism within the CNS. Furthermore, given the dys-regulation of glutamate in neurodegenerative diseases, GDH appears as a potent target for management of related neurological disorders.
We generated the Cns-Glud1-/- brain-specific GDH knockout mice by crossing Glud1 floxed (Glud1lox/lox) mice [10] with animals expressing the Cre recombinase under the control of Nestin cis-regulatory (Nes-Cre) sequence [11]. Heterozygous Cns-Glud1?/-were then crossed with Glu-d1lox/loxmice to obtain homozygous Cns-Glud1-/-, while
Cre was kept heterozygous [9]. In this specific study [9], animals were maintained on a mixed genetic background (C57BL/6 J 9 129/Sv) to avoid inbred strain-specific phe-notypes. Accordingly, floxed Glud1lox/lox littermates of Cns-Glud1-/-knockouts were used as control animals to optimize standardization of the genetic background between the two groups [9]. In other words, only Cns-Glud1 -/-knockout mice carried the Nes-Cre transgene. No gender differences were noticed.
The Cre transgenic lines represent invaluable tools to explore the role of a given protein in a specific tissue (conditional knockout), that can additionally be time-spe-cific (inducible Cre systems). Nevertheless, one should consider appropriate controls according to potential Cre-mediated effects. Some studies have reported side effects or phenotypes associated with Cre activity or transgene insertion [12–14]. For instance, the cause of haematologi-cal abnormalities after the systemic activation of CreERT2 described by Higashi et al. [15] was proposed to be the consequence of Cre-mediated genomic rearrangements. Such rearrangement within the mouse genome might hap-pen at pseudo-loxP sites, which have been shown to serve as substrates for Cre recombinase [15–17]. Some of the unexpected phenotypes associated with the presence of the Cre transgene might be due to the disruption of the genome loci where the transgene was integrated.
Another potential confounding effect of Cre technology is the misplaced recombination of the floxed target-gene. This is illustrated by some mouse models used to study insulin-secreting cells. In order to target Cre-mediated recombina-tion of floxed genes specifically in pancreatic beta-cells, Rip-Cre mice have been generated. These animals express the Cre recombinase under the control of the rat insulin (Ins2) promoter (Rip-Cre). However, infidelity of Rip-Cre transgene in the hypothalamus has been noticed in one transgenic line (Tg(Ins2-cre)25Mgn) [18], although not particularly in another one (Tg(Ins2-cre)1Herr) [19], potentially altering gene expression in nutrient-sensing neurons [20]. Such undesired hypothalamic effect might render the transgenic animals more likely to develop glucose intolerance under specific genetic backgrounds. Indeed, it was shown that perturbations in glu-cose homeostasis observed in a Tg(Ins2-cre)25Mgntransgenic line [18] can be accounted for by differences in genetic background [21]. Therefore, misplaced recombination com-bined with C57BL/6 J background might influence metabolic homeostasis and potentially contribute to a phenotype not fully specific for the beta-cell.
Some studies may have attributed roles to a target-gene, which were in fact partly or fully due to Cre toxicity. Limitation of Cre technology should not hide other possi-ble pitfalls, potentially more disturbing. Indeed, Cre-med-iated effects do not rule out alternative major confounding factors, primarily the genetic background, as discussed
Fig. 1 Abrogation of GDH in brain affects glutamate handling. a Simplified representation of glutamate fates in astrocytes and neurons (aKG, a-ketoglutarate; ALT, alanine aminotransferase; AST, aspartate aminotransferase; EAAT 1/2, excitatory amino acid trans-porters 1/2; GDH, glutamate dehydrogenase; Gln, glutamine; GLS, glutaminase; Glu, glutamate; GS, glutamine synthetase; SNAT 3/7, sodium-coupled amino acid transporters 3/7; vGLUT1, vesicular glutamate transporter 1). b Protein levels analysed by immunoblotting [9] of enzymes/transporters related to glutamate pathway in brains from CNS-specific Glud1 knockout (Cns-Glud1-/-) and Glud1lox/lox floxed control (C-floxed) mice. Actin (for GDH) and ezerin (for GS, EAAT2 and SNAT3) served as loading control. Values are mean ± SE, n = 2–5, * p \ 0.05 and ** p \ 0.01 using the Student’s t test
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above. Whenever possible, the genetic background should be homogenized between the studied groups [22], similarly to human studies based on twins [23].
Nestin-Cre mice are widely used to investigate gene deletion and cell function in the CNS. However, the Nes-Cre transgene itself was reported to induce a phenotype related to body weight gain. Indeed, it was observed that Nestin-Cre mice have smaller body weights [24], an effect not observed by others [25]. These apparently conflicting results have not been particularly highlighted to the scientific community, as they appeared as Supplementary Materials [24] and Addendum [25] to their respective papers. Maintained on a pure C57BL/6J background, Nestin-Cre mice purchased from the Jackson Laboratory (Origin, R. Klein, EMBL) display mild hypopituitarism [26]. Discrepancies between laboratories regarding body growth suggest that Nes-Cre transgene may render animals more susceptible to factors such as the genetic background or subtle environment specificities. This might have physiological consequences, in particular when Nes-Cre transgene is associated with the deletion of genes implicated in metabolic control. The
widespread use of Nestin-Cre mice has led to an increasing awareness of potential strain-dependent differences in body weight of these mice.
Given these strain differences, we further evaluated the Cns-Glud1-/- mice by comparing these conditional knockout animals with both Nestin-Cre controls carrying the Nes-Cre transgene (C-Cre) and Glud1-floxed controls lacking Nes-Cre but having loxP sites in the Glud1 gene (C-floxed). Immunobloting analyses showed comparable GDH expression in C-Cre and C-floxed brains as well as in non-excitable peripheral tissues known to express GDH (Fig.2a). Brain GDH enzymatic activity was not signifi-cantly different between the two transgenic control groups, i.e. C-Cre and C-floxed mice (Fig.2b). On the contrary, in brain homogenates of Cns-Glud1-/- mice we observed abrogation of GDH activity, both in the oxidative deami-nation and in the reductive amideami-nation directions. Wild type control (C-wt) mice exhibited brain GDH activity
Fig. 2 Expression and activity of GDH in Cns-Glud1-/- mice. aBrain-specific GDH deletion in Cns-Glud1-/-mice was assessed by immunoblotting. GDH expression in brains was compared with non-excitable tissues collected from Glud1lox/loxfloxed control (C-floxed), Nes-Cre control (C-Cre) and CNS-specific Glud1 knockout (Cns-Glud1-/-) mice. Actin served as loading control. Immunoblots are representative of 3 independent preparations. b Brain GDH enzymatic activity of C-floxed, C-Cre, Cns-Glud1-/-, and wild type control
(C-wt) mice measured in both oxidative and reductive directions [10], using glutamate (Glu) and a-ketoglutarate (a-KG) as substrates, respectively. Values are mean ± SE, n = 5–7, * p \ 0.05 and ** p\ 0.01 using one-way ANOVA
Fig. 3 Effects of Nes-Cre and Cns-Glud1-/-on body weight. a Body weights from Glud1lox/loxfloxed control (C-floxed), Nes-Cre control (C-Cre), CNS-specific Glud1 knockout (Cns-Glud1-/-), and wild
type control (C-wt) mice recorded over 16 weeks of life after birth. bIncreases in body weight for periods of 6 weeks measured over 2–8, 4–10 and 6–12 weeks of age. Values are mean ± SE; n = 7–27; * p\ 0.05, ** p \ 0.01 C-Cre versus C-wt; #p\ 0.05, ##p\ 0.01 C-Cre versus C-floxed, using one-way ANOVA
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comparable to the transgenic controls C-Cre and C-floxed (Fig.2b). Regarding body growth, we observed lower body weights associated with Nes-Cre transgene at the age of 7–8 weeks (Fig.3a) contributed by reduced weight gain during the weeks immediately following weaning (Fig.3b). The effect was transient and C-Cre animals could catch up body weight in comparison with the other transgenic groups by the age of 10 weeks when body growth stabilized. However, compared to wild type animals having more a C57BL/6J black 6 background, body weights remained slightly lower. Therefore, at the age of 10 weeks onwards, no significant differences in body weights were observed between transgenic mice maintained on a similar mixed genetic background, carrying or not Nes-Cre transgene; i.e. either C-Cre or Cns-Glud1-/-versus Nes-Cre-free C-floxed mice. According to these results, we favoured analyses of Cns-Glud1-/-mice at the age of 10 weeks onwards [9].
In conclusion, use of Nestin-Cre mice requires special attention regarding interpretation of body weight gain, in particular during the few weeks immediately following weaning. Pending appropriate controls and homogenization of the genetic background, Nestin-Cre technology is a valuable tool enabling disruption of genes of interest in the CNS. For instance, Cns-Glud1-/-mice lacking GDH in the CNS uncovered the fine tuning and compartmentalization of glutamate-glutamine handling in the brain.
Acknowledgments The author’s laboratory benefits from continu-ous support from the Swiss National Science Foundation and the State of Geneva. This study was also supported by a grant from the Syn-apsis Foundation.
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